Proteins have a central role in all biological processes including the development and treatment of human disease. Recent advances make it possible to isolate proteins of biological interest which are present in tissues in subpicomole quantities. Study of the structure function aspects of these proteins requires sequence information prerequisite to cloning and expression thereof. There is a consequential need for improved methods to sequence proteins and protein fragments at sample levels several orders of magnitude smaller than is now possible.
Currently, protein sequence analysis is primarily accomplished with the use of an automated sequencer using chemistry developed by Edman over 40 years ago (Edman, 1950) (FIG. 1). Since that time improvement in the instrumentation has resulted in the ability to sequence smaller and smaller sample quantities (mmole to pmol), although the original chemistry has remained essentially unchanged. Current automated instrumentation permits 10-20 cycles of sequence determination of 10-50 pmol of sample (Simpson et al., 1989).
The major limitation of Edman chemistry is the approximately one picomole practical detection limit of the PTH amino acids. The current method involves separation of the phenylthiohydantoin (PTH) amino acids by high-performance liquid chromatography (HPLC) followed by UV detection.
Various proposals to increase the sensitivity of the PTH's by use of radiolabeled, chromophoric, or fluorescent isothiocyanate reagents have been described. 4-(N,N'-Dimethylamino)azobenzene-4'-isothiocyanate (DABITC), a highly chromophoric reagent, was introduced by Chang (Chang et al., 1976). Fluorescent reagents, such as fluorescein isothiocyanate (Maeda and Kawauchi, 1968; Muramoto et al. 1984), and dansyl-containing isothiocyanates (Hirano and Wittmann-Liebold, 1986; Hirano and Wittmann-Liebold, 1987; Jin et al., 1986; Jin et al., 1989; Salnikow et al. 1987) have also been evaluated as sensitivity enhancing reagents. Synthetic amino acid analogues prepared using these reagents have shown subpicomole sensitivity by HPLC analysis. However, the use of these reagents in automated sequencing has not surpassed the sensitivity of the standard Edman methodology. It is postulated that the large chromophore of these reagents interferes with the derivatization and cleavage reactions of the Edman degradation. Radiolabeled reagents undergo autoradiodegradation which results in decreasing product yields and increasing amounts of labeled by-products.
An alternative method involves treatment of the anilinothiazolinone (ATZ) derivative normally formed in Edman chemistry with a fluorescent amine (Tsugita et al., 1989). The advantage is that the derivatization and cleavage reactions of the Edman chemistry remain unchanged. Theoretically this chemistry should permit sequencing on femtomole levels of sample. However, investigation has revealed a number of problems rendering this chemistry of little value toward the goal of more sensitive sequencing. Foremost is the instability of the ATZ-amino acids which are required for reaction with the fluorescent amine. The ATZ-amino acids, in particular the hydrophilic amino acids such as histidine, glutamate, and aspartate, rearrange to the PTH derivative so rapidly that reaction with the fluorescent amine was not possible. A possible solution to this problem is to convert the PTH amino acid back to the ATZ amino acid so that reaction with the fluorescent amine will be possible.
The aminolysis of PTH amino acids is discussed in detail by Pavlik et al. (1992).
Replacement of the fluorescent amine with a reagent such as N,N-dimethylethylenediamine (DMED) has been found by applicants to permit detection in the femtomole level using electrospray mass spectrometry. The introduction of the tertiary amine to the amino acid derivative was found to enhance detection of the amino acid by 25 times as compared to the PTH-amino acid, thereby making mass spectrometry a viable method for enhancing the levels of detection during protein sequencing. However, the use of DMED requires reaction with the ATZ intermediate and therefore tends to suffer from the same problems as the fluorescent amine approach.
Recently a new reagent, 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate has been introduced as a means of providing a thiohydantoin analogue with a quaternary amino group. (Aebersold et. al. (1992)) Although this reagent enhances detection of the released amino acid by mass spectral methods, it has a number of drawbacks. These include the synthesis of this reagent is complex, and the permanent positive charge makes this reagent polar and therefore requires the use of polar solvents to extract the amino acid derivatives. Such polar solvents cause sample washout thus limiting sequential yields. This problem necessitates covalent sample attachment with attendant poor yield therefore requiring larger amounts of sample and negating any potential benefit of enhanced sequencing capabilities. In addition, the large group used to attach the positive charge to the phenyl ring may have adverse affects on the kinetics of the sequencing chemistry. Dharmasiri, et al. synthesized Edman reagents having a pyridyl group as the basic site for enhanced detection of the TH amino acids which can be detected at the subfemtomole level by electrospray mass spectrometry.